Formation of covalent bonds between biopolymers by photoactivity plays an important role in researches of their structures and functions by providing important information to interaction between the molecules. The method is used as a tool for analyzing protein-protein interactions (Suchanek et. al., Nat Methods, 2, 261-7, 2005; Chou et. al., Chem Sci, 2, 480-83, 2011), protein-lipid interactions (Gubbens et. al., Chem Biol, 16, 3-14, 2009), protein-nucleic acid interactions (Pingoud et. al., Mol Biosyst, 1, 135-41, 2005), and the like.
Chemical conjugation of a photoactivatable group such as diazirine, benzophenone, and azide groups, to lysine residues of a protein has some disadvantages such as increase of hydrophobicity, interference of specific binding and increase of nonspecific binding.
In order to compensate such disadvantages, Jung introduced cysteine residues at specific positions of protein G and a photoactivatable group introduced to the cysteine residues of the protein G (Jung et. al., Anal Chem, 81, 936-42, 2009).
Studies to introduce nonnatural amino acids (NAAs) into proteins using in-vivo protein biosynthesis processes have been conducted (Davis and Chin, Nat Reviews Mol Cell Biol, 13, 168-182, 2012). Dr. Schulz's team in the US Scripps research institute conducted many studies in which in the natural world to introduce a NAA into the specific position of a protein by utilizing an amino acyl tRNACUA synthase and a tRNACUA pair recognizing TAG which is an amber stop codon, and thus, successfully, introduced various NAAs into proteins using in-vivo translation systems (Xie and Schulz, Nat Reviews Mol Cell Biol, 7, 775-82, 2006). Professor Tirrell's team of CALTECH in USA studied introduction of a NAA by preparing a methionyl tRNA synthase (MRS) variant. Particularly, azidonorleucine was successfully introduced into the methionine residues of dihydrofolate reductase of E. coli by inducing variants at Leu13, Tyr260, His301 sites (Crepin et. al., J Mol Biol, 332, 59-72, 2003) known as binding regions of the methionine of the MRS (Tanrikulu et. al., Proc Nat Acad Sci USA, 106, 15285-90, 2009).
Studies to introduce photoactivatable NAAs into proteins in the protein biosynthesis step in vivo have been conducted. Tippmann et. al. successfully introduced 4′-[3-(trifluoromethyl)-3H-diazin-3-yl]-1-phenylalanine (TfmdPhe) into a Z-domain protein in E. coli by using an amino acid tRNACUA synthase/tRNACUA system (Tippmann et. al., ChemBioChem, 8, 2210-14, 2007). Further, it was verified that lysine introduced with a diazirine group was successfully introduced into a target protein in E. coli and animal cell by using a pyrrolysyl (Pyl) tRNACUA synthase/PyltRNACUA system and a covalent bond between proteins by photoactivity may be induced (Chou et. al., Chem Sci, 2, 480-83, 2011). However, the residues have bulky and long carbon chains and thus, have a disadvantage of delicately forming a covalent bond with the target molecule. Such problems can be solved by using photoleucine (pL; L-2-amino-4,4-azi-pentanoic acid) or photomethionine (pM; L-2-amino-5,5-azi-hexanoic acid) which is very similar to leucine or methionine. Suchanek et. al. (Nat Methods, 2, 261-7, 2005) utilized the photoactivatable amino acid mimetic labeled in the protein in analysis of the bond between proteins after biosynthesizing the protein by adding the pM or pL photoactivatable amino acid mimetics in a medium without methionine or leucine in an animal cell culture. However, the inventors found very low protein expression in a minimal medium supplemented with pM instate of methionine in methionine-auxotroph E. coli B834 cells.
Since the antibody is very specifically bound with the antigen, the antibody has been used widely in medical researches associated with diagnosis and treatment of diseases and analyzing biological materials (Curr. Opin. Biotechnol. 12 (2001) 65-69, Curr. Opin. Chem. Biol. 5(2001) 40-45). Recently, as one immunological measurement method, a immunosensor of immobilizing the antibody to a solid support material and measuring current, resistance, a change in mass, an optical characteristic, and the like has been developed (affinity biosensors. vol. 7: Techniques and protocols). The immunosensor based on surface plasmon resonance using the optical characteristic has been commercialized, and a biosensor based on the surface plasmon resonance may provide qualitative information (whether two molecules are specifically bound with each other) and quantitative information (reaction kinetics and equilibrium constants) and detect them in real time without labeling and thus is particularly utilized for measuring binding of the antigen and the antibody (J. Mol. Recognit. 1999, 12, 390-408).
In an immunosensor, it is very important to selectively and stably immobilize the antibody to the solid support material. A technology of immobilizing the antibody largely includes a chemical immobilizing method and a physical immobilizing method. The physical method (Trends Anal. Chem. 2000, 19, 530-540) is almost not used due to low reproducibility and modification of the protein and the chemical method (Langumur, 1997, 13, 6485-6490) has been frequently used because the proteins are bound with each other well with a covalent bond to have good reproducibility and a wide application range. However, when immobilizing the antibody by the chemical method, the antibody is an asymmetrical macromolecule and thus the antibody loses orientation or loses activity of biding the antibody (Analyst, 1996, 121(3): 29R-32R).
In order to improve the antigen binding ability of the antibody, a supporter may be used before binding the antibody with the solid support material and a technology using the protein G as the supporter has been known.
The protein G as a protein of strong binding with most mammalian immunoglobulin G (IgG) Fc site is much utilized for preparing a highly sensitive chip with improved orientation of the antibody when preparing the antibody chip. Further, the protein G is bound with nanoparticles and the like and bound with the antibody to be utilized for preparing a target-oriented delivery system. However, since the binding of the protein G and the antibody is reversible, in the case of using a blood sample, the blood sample may be replaced with the antibody in the blood (Saleemuddin, Adv Biochem Eng Biotechnol, 64, 203-26, 1999), and thus it is important to form the covalent bond therebetween. The covalent bond between the molecules may be chemically induced, but has a disadvantage of causing a non-specific conjugation between the molecules.
Accordingly, the inventors made an effort to improve introduction of photomethionine (pM) in the biosynthesized protein and develop the protein G variant with improved antibody-specific to successfully prepare an E. coli methionyl tRNA synthase (MRS) variant capable of improving expression of a pM-introduced target protein in E. coli. Further, the plasmid encoding the MRS5m which is the MRS variant and the PG-C3 plasmid in which the positions of 32nd, 35th, and 40th of the third immunoglobulin G binding region C3 (PG-C3) of the protein G were substituted by Met residues and a position of 37th was substituted by an Arg residue were introduced into E. coli and purified to obtain the photoactivatable mimetic-introduced protein G variant, the specific covalent bond was formed by irradiating UV to the protein G variant and the antibody, and thus the photoactivatable methionine mimetic-introduced protein G variant may be utilized for preparing a highly sensitive biochip, a biosensor, and a cell-capturing chip, thereby completing the present invention.